Jul 1, 2010

Research Projects

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Developing a technique for the targeted integration of genes in plants

(2008 – 2011) University of Karlsruhe, Botanical Institute, Chair of Molecular Biology and Plant Biochemistry

Topic

When inserting new genes in plants, it is impossible to predict where in the genome integration will occur. As a result, the newly inserted gene could impair the functioning of other genes. Targeted insertion at known and well-defined sites in the genome could eliminate this uncertainty.

The aim of this project is to further develop a technique for the targeted integration of a transgene at any chosen site in the plant genome (gene targeting). This technique is based on homologous recombination, a naturally occurring mechanism whereby genetic material rearranges itself. As yet, gene targeting techniques developed for plants remain inefficient, i.e. the successful integration of the transgene at a specific site in the plant genome occurs too infrequently.

In the model plant Arabidopsis thaliana three genes (BRCA1, BRCA2 and BARD1) have been identified, the absence of which leads to significantly fewer homologous recombination events occurring. The aim of the experiment described here is to increase the number of homologous recombination events through the over-expressionof this gene. If this is successful, tests will be conducted using two different gene targeting systems to determine whether this makes the technique more efficient.

Precursor project:

Further information on methods:

Experiment description

In the first stage, Arabidopsis plants containing the three named genes will be transformed (cf. transformation), both individually and in different combinations. Over-expression of the genes will be induced by selecting suitable control elements (promoters). Molecular-biological methods will be used to investigated the sites in the plant genome at which the genes have been inserted. A colour reaction will be used to identify which genes or gene combinations result in an increase in homologous recombination events. These plants or gene constructs will then be used in further experiments to investigate whether it is possible to improve the efficiency of the gene targeting technique in two different test systems.

In the first system, the selected plants will be transformed with a further gene construct which carries a reporter gene. The reporter gene is flanked by sub-sequences of a gene which is found in the genome of A. thaliana and is expressed only in the seed. The reporter gene will be integrated at the site where this gene occurs in the plant genome via homologous recombination and, as a result, it will also be expressed in the seed, and nowhere else. If gene targeting is successful, the plants will produce red fluorescent seeds, which can be very easily identified under a fluorescent microscope and investigated further.

The second gene targeting system to be investigated was developed in the precursor project. Two plant strains will be genetically modified and crossed with each other. The genome of one strain contains a known and well researched target site for the integration of a transgene. The genome of the other strain contains the transgene, flanked by DNA fragments whose sequences are homologous to those of the target site. When the plants are crossed, the transgene should become integrated at the target site via homologous recombination, and this can be verified using a reporter gene and an antibiotic-resistance gene. In the precursor project, however, this integration was ultimately not demonstrated.

The two parent strains from the precursor project will then be transformed with the gene constructs from the first part of the experiment, which successfully brought about an increase in homologous recombination events. Then the two strains will be crossed again to test whether the transgene is integrated more efficiently than in the precursor project.

Results

Gene constructs were produced with different promoters (ubiquitin promoter or double 35S promoter) which were designed to cause an over-expression of the BRCA1 and BARD1 genes. The constructs also contained antibiotic-resistance genes. These constructs were used to transform Arabidopsis plants. Subsequent selection on a culture medium containing antibiotics (PPT) enabled the identification of several transgenic lines. These were propagated within their own lines so that the progeny could be reselected individually on the culture medium. The aim was to obtain progeny that contained the transgenic DNA at only one site in the genome. The idea was to prevent undesirable effects, such as silencing of the transgene.

The progeny of the transformed plants were unable to grow on the culture medium containing PPT. The resistance gene had evidently been silenced. This is thought to have been caused by too many 35S promoters in the plant gene. Transgenic Arabidopsis lines are currently being produced that contain far fewer 35S promoters. Initial segregation analyses show that the silencing of the marker gene observed previously no longer occurs.

The aim was also to produce a gene construct that triggers an overexpression of the BRCA2 gene in Arabidopsis. This part of the project is not yet complete because of experimental difficulties.